Numerous viral vectors have been developed with the ultimate goal of efficiently delivering replacement genes to human organs, without causing damage to the targeted cells. However, the luminal cells of the human airway epithelium are generally refractive to most vectors. Respiratory viruses like respiratory syncytial virus (RSV) are unique in that they specifically target these cells. Typical infection causes cell death after several days however it is now possible to alter RSV in such a way that it may no longer be cytotoxic. As a result, RSV could be a useful vector for delivering a transgene, such as the CFTR channel, to the respiratory epithelial cells of cystic fibrosis patients. We now know that RSV specifically infects the ciliated cells in primary cultures of human well-differentiated airway epithelial (HAE) cultures developed by the PPG cell culture core, and is cytotoxic on these cells over the course of a week. In this proposal, a number of reagents will be further developed for airway specific gene delivery. Specifically, attenuated vaccine candidate strains of RSV will be tested for lack of cytotoxicity in HAE cells. In addition to attenuated RSV strains, recent work with RSV 'replicons' that lack the viral glycoprotein genes and therefore are unable to spread are now available for further vector development. To generate infectious vector from these replicons, the viral glycoproteins will be provided from integrated gene copies in an inducible manner similar to retroviral packaging systems. Non-replicating RSV also provide the opportunity to deliver controlled (dose dependent) amounts of the CFTR transgene which may be enough to correct a genetic defect such as cystic fibrosis. RSV vectors capable of producing the highest amounts of vector in producer cells and the lowest cytotoxicity in target HAE cells will be engineered to carry CFTR transgene. Unique to this PPG is the ability to test resulting RSV-CFTR vectors in HAE cultures derived from cystic fibrosis patients for repair of bioelectric defects, restoration of airway surface liquid depth, and the restoration of mucociliary clearance.